Introduction

Cotinine derives from nicotine that in turn, derives from the dried leaves of Nicotania tobacum. Once absorbed in the body, nicotine rapidly metabolizes to cotinine and other metabolites and cannot be detected in significant quantity in the urine. Cotinine, on the other has a much longer half-life and is the principal urinary metabolite. The cotinine EIA kit can be used to determine quantitatively the concentration of cotinine in the urine sample.

Principle of Cotinine Determination

The enzyme immunoassay for cotinine is based on the competition between the cotinine to be assayed and the Cotinine-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against cotinine, coated onto microwells. The sample containing the cotinine, and the Cotinine-Alkaline Phosphatase conjugate, when added to the microtiter wells, compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed in order to remove non-bound components. The bound enzymatic activity is then measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of cotinine in the sample. The concentration is calculated on the basis of a standard curve.

Reagents

• 96-well microtiter plate. Twelve strips of 8 detachable wells, coated with Anti-Cotinine antibody.
• One vial of 0.9 ml Negative Calibrator containing 0 n/ml of cotinine.
• Three vials of Positive Calibrator (0.75 ml) containing 50 ng/ml, 200 ng/ml and 750 ng/ml of cotinine.
• One bottle containing 10.5 ml of Cotinine-Alkaline Phosphatase (CTN-ALP) conjugate.
• One bottle containing 10.5 ml of p-Nitrophenyl Phosphate (pNPP) substrate.
• One bottle containing 15 ml of 10xWash Buffer (PBS-Tween).
• One bottle containing 5.5 ml of Stop Solution, 3N NaOH.

Assay Procedure

• 25 μl of standard or sample to the bottom of each well.
• 100 μl of CTN-ALP conjugate to each well. Incubate at room temperature for 40 minutes.
• microtiter wells 3 times with wash buffer.
• 100 μl of pNPP substrate to each well. Incubate at room temperature for 20 min.
• 50 μl of stop solution to each well.
• absorbance at 405 nm using ELISA reader.

Cotinine Inhibition Curve